TODAY

1
TODAY

• Assignments
• Collect Hamburger Report, Pre-lab 5.2
• Return Pre-lab 5.1
• Exercise 5.2
• Determine Biochemical test results
• Environmental isolate
• Continue stains
• Facultative anaerobes
• Intro to Exercise 6

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McMillan Ch. 8 Mechanics and Technicalities

• Use the scientific names of organisms.
• Common names are not universally agreed upon
• May vary with location, language, etc.
• Each species has ONE scientific name
• Includes the genus and species
• Typically Latin
• May also include the name of the first person to
  publish about it- You dont need to include this
• In microbiology, the name may also include a
  strain, which is generally included

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McMillan Ch. 8 Mechanics and Technicalities

• The genus and species are always italicized
• Strains are not italicized
• Give the full name the first time you refer to
  the organism
• Abbreviate later in the text
• If the abbreviation falls at the beginning of a
  sentence, spell it out

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McMillan Ch. 8 Mechanics and Technicalities

• Do not use an article immediately before the
  scientific name
• The, a, an
• Do not pluralize scientific names
• The genus must always precede the species
• Never use just the species name
• The genus may be used without the species to
  refer to a taxonomic group

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McMillan Ch. 8 Mechanics and Technicalities

• Taxonomic groups above the genus are capitalized,
  but not italicized
• It is common for scientists to modify the ending
  the Latin names to create a common name
• These are not capitalized or italicized

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McMillan Ch. 8 Mechanics and Technicalities

• Other common names are also not italicized or
  capitalized
• There are exceptions
• Places and names ex. English ivy
• Standardized names ex. Chipping Sparrow, but not
  sparrow
• Undesignated species
• Common problem in microbiology
• Use the genus and sp. to abbreviate species
• Ex. Bacillus sp.

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McMillan Ch. 8 Mechanics and Technicalities

• Record time according to a 24-hour system.
• Dont use AM or PM
• Use symbols and abbreviations commonly used in
  biology.
• McMillan lists many common abbreviations
• Note the most are not followed by a period

8
McMillan Ch. 8 Manuscript Format

• The way your paper looks-the formatting- is the
  first impression for the reader
• Journals normally have strict formatting
  guidelines, the paper will be rejected if you
  dont follow them!

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McMillan Ch. 8 Manuscript Format

• Paper, margins, and spacing.
• Paper 8 1/2 by 11 in. white paper (a.k.a
  standard printer paper)
• Use the default setting for margins and tabs in
  word
• Generally double spaced

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McMillan Ch. 8 Manuscript Format

• Title Page
• Title, name, course information, date
• Information should all be centered
• The title should be about one-third down the page
• Single spaced if it has multiple lines
• Do not underline, italicize, or use quotations
• Capitalize all important words
• All other info is below the title, double spaced

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McMillan Ch. 8 Manuscript Format

• Pagination
• Number pages in the upper right-hand corner
• Use only Arabic numerals (1,2,3, etc.)
• Do not number the title page
• Each section does not need to be on a separate
  page
• Use default page settings in word

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McMillan Ch. 8 Manuscript Format

• Headings
• Use headings and subheadings sparingly.
• Generally need no more than two levels of
  headings
• Can fragment your paper
• Make headings informative and concise
• Ex. Results, Stains, Biochemical Tests

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McMillan Ch. 8 Manuscript Format

• Use the same format for all the headings of one
  level, and a different format for headings of the
  nest level
• Be consistent!
• Use the same grammatical form for all headings
• Ex.
• Verb Measuring spatial variations of infection
• Noun Spatial variations of infection

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McMillan Ch. 8 Manuscript Format

• Figures and Tables
• McMillan goes over several common ways to
  incorporate figures and tables
• How I want it
• Tables and figures should be in the text, after
  the paragraph where they are first mentioned
• Titles to tables and Figures should be abvoe the
  table or figure.

15
Carbohydrate Fermentation

• The ability to ferment following carbohydrates
  glucose, sucrose, lactose and mannose.
• Tests for gas production color change due to pH
  change.
• Phenol red changes to yellow when conditions are
  acidic-when acidic, sugar is being fermented.

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Carbohydrate Results
Negative orange or red
Positive yellow, or yellow with gas
17
Nitrate Reduction Test

• p. 68-70 photographic atlas
• Some microorganisms that usually use molecular
  oxygen as a terminal electron acceptor can
  substitute nitrate (NO3-) for this purpose under
  anaerobic conditions (e.g., Pseudomonas).
• Nitrate can be reduced to nitrite (NO2-) and some
  microorganisms can reduce the nitrite further to
  ammonia (NH3) or even to nitrogen gas (N2).

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Nitrate Reduction 24/48 hour check

• Check for the presence of gas in the Durham tube.
  
• If there is gas in the Durham tube, it is
  nitrogen and this observation alone is a positive
  test for nitrate reduction.

Negative ? Go on to next step
Positive ? Stop
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Nitrate Reduction

• Step 2 Gas Present ? Do Following
• To refrigerated tube, add 10 to 15 drops of
  Nitrite A reagent.
• To same tube, add same amount Nitrite B reagent.
• Note that dimethyl-alpha-naphthylamine is closely
  related to compounds that are carcinogenic.
• If any of this reagent contacts your hands, wash
  them immediately.

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Nitrate Reduction

• Positive - If the culture turns red within 15
  min. it is positive for the presence of nitrite
  and positive for nitrate reduction.
• Negative - If after 15 min. there is no color
  change, then one of two events have occurred
  either the nitrate has not been reduced or
  nitrate has been reduced beyond nitrite to
  ammonia or nitrogen gas. ? Perform next test!

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Nitrate Reduction

• Step 3 No change after Nitrate A and B were
  added
• Add small amount of zinc powder to test tube.
• Red color within 15 min test is positive for
  presence of nitrate, but negative for nitrate
  reduction.
• No color change nitrate has been reduced to
  either ammonia or nitrogen gas and is positive
  for nitrate reduction.

22
Nitrate Reduction Procedure
23
Motility Test

• p. 67-68 photographic atlas
• True motility (directed movement) is different
  than Brownian movement. Brownian movement is
  caused by invisible molecules striking the
  bacteria making them appear to vibrate rather
  than the bacteria actually moving from one place
  to another.
• Motility can be observed in a wet mount or
  hanging drop preparation of the organism.
  However, wet mounts tend to dry out quickly
  rendering the organisms immotile.

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Motility Test Results
Positive Cloudiness in whole tube
Negative Cloudiness only around stab mark
25
Simmons Citrate

• p. 51-52 photographic atlas
• This test determines if an organism can transport
  citrate and use it as the sole carbon source.
• Organisms that live in this medium can also use
  ammonium ions (instead of amino acids) as the
  sole nitrogen source.
• The pH indicator is brom thymol blue. This
  indicator is green at neutral pH but turns blue
  above pH 7.6.
• When citrate is being utilized, bacteria increase
  the alkalinity of the solution.

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Simmons Citrate Results
Positive color changes from green to blue
Negative - No color change
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Urea Hydrolysis

• p. 79 photographic atlas
• Urea is a common metabolic waste product that is
  toxic to most living organisms.
• Urease is an enzyme that hydrolyzes urea into
  ammonia and carbon dioxide.
• This test differentiates organisms by their
  ability to hydrolyze urea.
• If urease is present, ammonia will be released
  and the pH will rise.

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Urea Results

• Positive -
• Cerise (a hot pink cherry color)

• Negative Yellow, Orange, or light pink

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Kligler's Iron Agar

• p.61-62 photographic atlas
• Kligler's iron agar is used to test for the
  production of hydrogen sulfide (H2S) gas.
• The production of H2S often results from the
  deamination of the sulfur containing amino acid
  cysteine.
• This medium contains ferrous sulfate, which
  reacts with H2S to form a dark precipitate of
  iron sulfide.

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Kligler's Iron Agar

• A positive test shows a dark precipitate that has
  formed in the tube. The absence of a precipitate
  is a negative test.
• Since this medium also contains glucose, lactose
  and phenol red, the medium might also turn yellow
  due to the fermentation of these carbohydrates.
• Note that a yellow color in the tube without a
  dark precipitate is still a negative test for H2S
  production.

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Kligler's Iron Agar Results
Negative - no precipitate, any yellow, red, or
orange color without a precipitate
Positive dark precipitate
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Gelatinase Test

• p. 59 photographic atlas
• Gelatin is a heterogeneous mixture of very large,
  water-soluble proteins and is prepared from
  collagen by boiling skin, tendons, ligaments,
  bones etc., with water.
• Many microorganisms produce an enzyme called
  gelatinase that can degrade or breakdown the
  gelatin into smaller polypeptides and amino acids
  that can be taken up and used by the cell.
• Gelatin liquefies at temperatures above 30?C but
  solidifies at 4?C. When hydrolyzed by the enzyme
  gelatinase, however, gelatin does not gel when
  placed at 4? or 5?C.

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Gelatinase Test Results

• After one week incubation, chill the tubes in the
  refrigerator.
• Do not shake the tubes when transferring them to
  the ice bath as this medium is already a bit
  "loose."
• Negative - Gelatin tube should "firm up" when
  chilled.
• Positive - If your unknown organism produced
  gelatinase and hydrolyzed the gelatin, the
  gelatin will remain liquid after chilling.
• If your unknown organism did not hydrolyze the
  gelatin after one week incubation, continue
  incubating your unknown for another week.

34
Gelatinase Test Results
Positive Gelatin remains liquid after chilled
Negative Gelatin is firm after chilled
35
Starch Hydrolysis

• p. 75-76 photographic atlas
• Starch is a complex polysaccharide that can be
  hydrolyzed by a variety of microorganisms via
  extracellular enzymes called a-amylases.
• Starch molecules are much too large to be taken
  into the cell, and must be broken down into their
  constituent parts just like large proteins are.

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Starch Hydrolysis Results

• Refrigerate after 24 48 hours.
• Add a few drops of Gram's iodine (i.e., use just
  enough to cover the surface of the plate).
• Areas on the plate that contain starch will form
  a dark blue or purple complex. Areas around
  colonies in which the starch has been hydrolyzed
  will appear as clear zones.
• Positive - A clear zone around your test organism
  after treatment with Gram's Iodine.

37
Starch Hydrolysis Results
Positive
Negative
38
Casein Hydrolysis

• In order for microorganisms to take advantage of
  the carbon and nitrogen in large proteins found
  in their environment, the proteins first have to
  be broken down into individual amino acids or
  small peptides (chains of a few amino acids) in
  preparation for transport into the cell.
• The cell accomplishes this by excreting
  extracellular enzymes called proteases which
  break down proteins in the environment.

39
Casein Results
Positive zone of clearing around organism
Negative no zone of clearing
40
Lipid Hydrolysis

• p. 62-63 photographic atlas
• Lipases (or esterases) are enzymes which
  hydrolyze the ester linkages that hold fatty
  acids to glycerol.
• Positive zone of clearing around organism.
• Negative no zone of clearing (lightening of the
  medium is also a negative result)

41
Lipid Hydrolysis Results
Positive zone of clearing around organism
Negative no zone of clearing around organism
42
Facultative Anaerobes

• Many bacteria can grow both aerobically and
  anaerobically. Organisms that can grow in the
  presence or absence of oxygen are call
  "facultative anaerobes" (E. coli is an example).
• To determine if your unknown organism is a
  facultative anaerobe, inoculate a TSA plate with
  your unknown and place it into the anaerobic jar
  that your instructor has prepared.
• The oxygen will be removed chemically and the
  organisms allowed to incubate until the next
  laboratory period.

43
Environmental Isolate

• In the time remaining, continue microscopic
  examination of your environmental unknown(s),
  including methylene blue, Gram stains, capsule
  stain, acid-fast, and endospore stain.
• Examine to determine best storage conditions.

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Identifying Your Unknown

• Every observation you have made up to this point
  could be important
• Always verify any observations you make
• Use controls for every test
• Never go forward in the flow chart if you have
  any doubts
• Use common sense and reason

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Identifying Your Unknown

• Bergeys can help you determine the possible
  identification of your environmental unknown only
  if..
• all your test results are correct
• you perform all the right test
• you use your head and constantly review what you
  know for sure and what you need to find out to go
  forward

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Identifying Your Unknown
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Identifying Your Unknown
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Identifying Your Unknown
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Bergeys Manual

• After you have determined your unknowns
• Morphology
• Arrangement
• gram reaction
• O2 requirements
• evaluated your biochemical results
• Bergeys can be very useful

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Bergeys Manual

• Bergeys contains many
• useful tables to help you
• determine systematically
• the family, genus and species
• of your unknown.
• But it is not only tables..

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Bergeys Manual
Bergeys is an extensive resource of information
on thousands of bacteria But you have to
take the time to read it to get the most out of it
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Bergeys Manual
Learn to use the table of contents and index to
locate specific information Such as Section 12
Gram Positive Cocci
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Identifying Your Unknown

• So do it!
• Continue microscopic examination of your
  environmental unknown using wet mounts and simple
  and differential stains
• Check TSA slants to determine your organisms best
  storage conditions.
• Grab Bergeys and start looking

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Exercise 6

• Bacterial Growth

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Bacterial Growth

• Bacterial Cell Growth is the increase in the
  quantity of all cellular structures and
  components. This increases the cell's size until
  division occurs.
• A vegetative cell is a cell that is actively
  growing and dividing.
• Microbial Growth is measured by assaying the cell
  number or population of cells in mass.

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Bacterial Growth

• Generation Time (or doubling time) is the time it
  takes an individual cell to divide or for a
  population of cells to double.
• Bacterial growth is logarithmic (exponential) - 2
  cells divide into 4, 4 cells divide into 8, 8
  cells divide into 16, etc.

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Bacterial Growth Phases of Growth

• Lag Phase
• No significant increase in number of cells
• Metabolically active
• Growing in size
• Synthesizing enzymes
• Taking up molecules from the media

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Bacterial Growth Phases of Growth

• Log Phase
• Population grows at an exponential rate
• Generation time can be calculated
• Generation time time it takes for the
  population to double
• A genetically predetermined interval

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Bacterial Growth Phases of Growth

• Stationary Phase
• Number of new cells produced is equal to the
  number of cells that die
• Nutrients may become limiting
• Toxic waste materials may be accumulating

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Bacterial Growth Phases of Growth

• Death Phase
• Number of live cells decreases exponentially
• Medium is not supportive of cell division
• Cells can take on strange shapes

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Bacterial Growth Enumeration methods

• Hamburger (viable count) exercise
• Direct counting method
• Viable count assays (using plate counts)
• Bacterial population at one point in time
• Bacterial growth exercise
• Indirect counting method
• Measure turbidity (using a spectrophotometer)
• Bacterial population growth over time

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Bacterial Growth Questions

• How does glucose affect growth rate?
• How do aa and peptides affect growth rate?
• What is the generation time of a culture in
  minimal media?
• What is the generation time of a culture in
  supplemented media?

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Bacterial Growth Minimal Media

• Measure E. coli ML30 growth rate in glucose
  minimal medium
• Glucose minimal medium contains
• Glucose
• Salts

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Bacterial Growth Minimal Media

• Glucose will serve as
• sole carbon source
• sole energy source
• Cells must make everything else they need for
  growth.
• amino acids, proteins, carbohydrates, lipids,
  nucleic acids, and vitamins

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Bacterial Growth Nutritional Shift-Up

• We will add 10 yeast-extract peptone (YEP) to
  the medium.
• How will the growth rate change after the media
  is supplemented?

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Bacterial Growth Nutritional Shift-Up

• Yeast-extract digest of yeast, provides a good
  general base for culture media, components are
  undefined
• Peptone proteinaceous materials (meat, soy
  beans, etc.) digested by enzymes or acids
• Agar complex polysaccharide that serves as a
  solidifying agent (seaweed)

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Bacterial Growth Optical Density

• We will be using an indirect method of measuring
  bacterial population the spectrophotometer.
• The spectrophotometer uses the turbidity of the
  culture to determine the amount of light that is
  deflected off the cells by measuring the optical
  density (OD) of the culture.
• The changes in absorbance are due to the light
  scattering off the cells (99 of which are viable)

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Bacterial Growth Optical Density

• The greater the number of organisms, the more
  light they deflect from the photodetector.
• A cloudier culture, with more growth, will have a
  higher OD then a less cloudy culture, since more
  light is deflected by the denser culture.

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Bacterial Growth Optical Density

• Higher cell concentration
• More light is scattered
• Higher optical density
• (greater absorbance value)
• (lower transmittance value)

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Bacterial Growth Procedure

• The E. coli ML30 will be in 0.0025 glucose to
  begin
• You add 0.5 ml of .20 to start growth
• This is time 0
• Monitor the growth over 20 min intervals
• get 3 points and plot the glucose line

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Bacterial Growth Procedure

• Now perform a nutritional shift up
• Calculate remaining volume
• Calculate amount of YEP need for 0.5 conc..
• Remember C1 x V1C2 x V2 ?
• (10) x (ml of yep needed) (ml of culture left)
  x (0.5)
• Add required YEP to flask
• This is new time 0
• Monitor the growth over 20 min intervals
• get 3 points and plot the YEP line

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Bacterial Growth Procedure

• From the graph determine the doubling time for
  the glucose and YEP plots
• Doubling time generation time(g)
• This is a direct comparison of growth in a
  defined minimal medium versus a complex medium

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Bacterial Growth Procedure
The Spectrophotometer
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Bacterial Growth Procedure

• Please dont adjust the controls on the
  spectrophotometer
• We will zero it with a blank before you take your
  readings
• Use the same cuvette throughout the experiment
• Rinse it after every sample with DI
• set it upside down in the rack provided to drain
• Dump everything into the beaker provided
• Dont let it fill up to full

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Bacterial Growth
Plotting your data
OD550

• Determine your Y-axis scale

0.10

• Determine your X-axis scale

• Label your axes

• Plot your points as you go

• Draw your best fit line

• Determine your growth rate

0.010
0
20
40
60
80
100
120
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Step by Step
50 ml aliquots in 125 ml flask with group labels
E. coli .0025 glucose in Fernbach Flask
Record initial OD 0.012-0.020
Shaker Bath (turn back on after sampling)
Groups stagger start times at 3 min intervals
Determine your doubling time and cleanup
Repeat 2-3 X 20min intervals and plot as you go
Calculate the YE-P needed for 0,5, add YE-P to
flask and take YE-P Time 0
Store your cuvette upside down in the rack
OSYL Waste
Repeat 2-3 X 20min intervals (record your exact
times) and plot your data as you go
Dump the culture and the rinse into the waste
beaker
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Next Week

• Assignments
• 1st submission Hamburger Report returned
• Pre-lab 6 due
• Exercise 6
• Spectrophotometric Determination of Bacterial
  Growth
• Work on Environmental Isolate
• request specialized tests